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Microbial Communities & Feeding Specialization
of Insects at The Sevilleta LTER
Dan Colman
Focal Organism – Epicauta longicollis
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Member of Meloidae family
18-25 mm in length, slender, elongated body form
Found throughout desert southwest & Mexico
Emerge in early summer
Occur in swarms, feed
on varying flowering
plants
• Predominantly feeds
on Solanum elaeagnifolium
at the Sevilleta LTER
(Schafer & Toolson, 2007)
Solanum elaeagnifolium
(silverleaf nightshade)
• Perennial weed native to the western U.S. & S. America
• 0.3 - 0.9m in height
• Very low soil quality & water requirements – adapted to semi-arid
environments
• Contain varying levels
of alkaloid neurotoxin, solanine,
throughout plant
– Problematic for ranchers
– 3-6 mg/kg body weight can lead
to fatality
• Grazing is very minimal
– E. longicollis only known grazer
at the Sevilleta LTER
Previous Research On
E. longicollis
• Behavioral studies recently conducted at the Sevilleta
LTER by Schafer & Toolson
• Investigation into the feeding behavior of E. longicollis
• Strong evidence seen for feeding preference on varying
plant parts
• Specialization seen in feeding indicates adaptation to
feed predominantly on a plant of known high toxicity
Objectives For The Study
• Characterize the microbial community residing within E.
longicollis
– Compare against microbial communities within other
insects of same region (various feeding habits)
– Analyze microbial communities over time intervals for
changes in structure
– Analyze intraspecific community variation
• Determine if microbial community growth on S.
elaeagnifolium media mirrors E. longicollis feeding
preference
Hypotheses
• Hypotheses:
Specialized feeders like E. longicollis will have a
more specialized microbial community than nonspecialized feeders.
Microbial growth quantities & species richness on S.
elaegnifolium media will mirror E. longicollis feeding
preference
Analysis between individuals of the same species
will show less variability than analysis interspecifically
Justification For Research
• To adequately understand natural history of insects – an
understanding of endemic microbial communities is necessary
• Contribute to the understanding of co-evolution between microbial
communities & their hosts
– E. longicollis’ specialized diet may indicate the presence of a
novel microbial strain/organism in microbial community
• Add evidence for hypotheses
about microbial dispersal
among distinct “island” habitats
http://www.jgi.doe.gov/education/bioenergy/bioenergy_4.html
Environmental Sampling – A genetics
Based Approach
• Cultivation is inherently biased to a few types of
microorganisms, often not the most important
– Useful as a supplement to a genetics based approach
• Estimated that a very small amount of bacteria are
culturable
• The most effective technique available for sampling
microbial communities
• Allows us to take a snapshot at a specific time of
community
Environmental Sampling – A Genetics
Based Approach
Community Sample
Total Community DNA
Selection & Amplification
of Target Genes
Insertion of Target
Genes Into Vector
Vectors Taken In By
Live E. coli Cells
E. coli Colonies Grown,
Samples Selected at random
Data Analysis
Each Culture’s Plasmid (Vector) DNA Harvested & Sequenced
Other Species Sampled
Pasimachus elongatus
Omorgus scutellaris
-9-15 mm in length
- 21-28 mm in length
- Found from plains of S.W. north to
Canada
- Feed on smaller invertebrates
- Found in southern U.S. & Mexico (arid
areas)
- Associated with scat, particularly coyote &
owl
- Detritivores (fur, skin, feathers in scat)
Other Species Sampled
Trimerotropis pallidipennis
-25-45 mm in length
- Found from British Columbia to
Mexico
- Generalist feeder (grasses and forbs)
Eleodes longicollis
- Common darkling beetle of the desert
southwest
- Feed on bits of seeds & vegetation
(associated with Pogonomyrmex
rugosus ants)
- Defensive secretions that give them
the “stinkbug” pseudonym
Other Species Sampled
Cibolacris parviceps (cream grasshopper)
-20 – 32 mm in length
- Found throughout the S.W. U.S.
into northern Mexico
- Thrives in desert habitats
- Feeds on creosote bush
Study Sites
• All insects sampled from two areas within close proximity
(~6 km) and of similar vegetative growth
Sampling Methods
• Each species sampled from one of the two site locations
• 3 individuals initially sampled for all 5 comparison
species – 1 individual captured every week for two
weeks
– Epicauta sampling differed
• Samples cooled upon capture
– Frozen upon returning from the
field
Dissection/Preparation for DNA
Extraction
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Dissections were performed using sterile technique
Performed as soon as possible upon return from field
Dissection protocol identical for every sample
Entire intestinal tract prepared for sampling by adding to
a phosphate buffer solution
DNA Extraction
• Bead beating step first used to help bring sample tissue into a
useable solution
• Chemical process employed using standard DNA extraction protocol
• Cells in solution lysed (CTAB)
• Free proteins digested
(Proteinase K)
• Cell membranes pulled apart
(SDS Detergent)
• DNA in solution separated by
extracting DNA into distinct
layer (Chloroform)
• DNA precipitated out of solution
into a pellet form
Pure DNA
Polymerase Chain Reaction
• Enables us to select for one gene & amplify quantity of that gene
“found” in solution
• Reaction targets 16s rRNA gene – conserved across microbial
lineages
• A reaction mixture is made
with all the necessary reagents
for target genes to be
duplicated according to
standard PCR protocol (primers,
polymerase, nucleotides, sample
DNA, reaction buffer)
– Universal primers used
Polymerase Chain Reaction
• Reaction mixtures are first heated
– Allows DNA double strands to separate
– Then cooled – Primers attach at target gene ends
• Reaction then slightly heated again - polymerase
attaches
– Polymerase attaches free nucleotides to template at primer 3’
end
• Gene duplicate copies are exponentially made with
every full cycle (a total of 30 cycles) – potentially many
16s rRNA genes representing many different microbes
can be amplified hundreds of thousands of times
• PCR is a sensitive process – troubleshooting often
required to have a successful reaction
Monitoring For Positive Results
• PCR products analyzed before moving forward
• PCR reaction products
ran on agarose gels
• Gels analyzed for
fluorescence at 15001600 base pair level
which indicates presence
of 16s rRNA in reaction
product
• Successful reactions
saved for processing
Inserting Target Genes Into Vectors
• Target genes must be isolated & separated from one
another
• Insert each target gene into a “cloning vector”
– Each copy obtained from PCR now isolated from one another
– “Housed” individually in vectors
• Reaction with PCR
product & vectors
• Upon incubating the reaction at room
temperature, our target gene is taken
in by the vector
Vectors Taken Up By Live
Escherichia coli
• E. coli possess the ability to uptake free DNA from
environment
• Reaction promotes uptake of vectors (with target genes
now inserted)
– Each E. coli cell represents one of our target genes
• Cells cultivated & colonies formed
– Each colony will contain identical copies of vector within all cells
of colony
• 96 colonies will be selected at random per
each insect sample
http://strubiol.icr.ac.uk/extra/baculovirus/bact_protocols.html
Vector DNA Harvested From E. coli
• Plasmid DNA is harvested from colony samples
– Similar process to DNA extraction initially performed
• Each colony’s DNA then sequenced
• All 96 sample’s sequences ran through National Center
For Biotechnology’s Database of known sequences
– Nucleotide sequence similarity/distance indicates
relatedness to known species
• 96 sequences per insect sample now available for
analysis
http://biosci.sierracollege.edu/resources/bio-teaching/pcr-id.html
Environmental Sampling – A Genetics
Based Approach
Community Sample
Total Community DNA
Selection & Amplification
of Target Genes
Insertion of Target
Genes Into Vector
Vectors Taken In By
Live E. coli Cells
E. coli Colonies Grown,
Samples Selected at random
Data Analysis
Each Culture’s Plasmid (Vector) DNA Harvested & Sequenced
Genetic Sampling Supplement:
Cultivation Experiment
• 6 teas were made using Solanum elaeagnifolium plant
tissue
• 3 types of plant tissue – 2 treatments
• Concentration of tissue/water constant for all 3
• Tea medium filter sterilized
• Inoculated with pool
of Epicauta intestinal
material
• Incubated for 9 days
• Samples to be
genetically identified
Preliminary Observations
• Preliminary microscopy
• All 3 medium types look similar
– Cell counts seem to be low
– Predominantly a few shapes seen
• Genetic sampling of all 6 treatments necessary for
conclusions to be made
Future Data Analysis
• Each sequence represents one data point (one microbe
individual originally in sample)
• Rarefaction Curves with unique sequences
• Cluster Analysis: Arrange sequences into phylogenetic
trees (per each sample, all samples total, per each
species, etc.)
• Significant differences in community structure can be
analyzed between
1) Individuals of the same species
2) All species
3) Species of different feeding behavior (specialized/nonspecialized)
4) Different collection time points
Mesak et al. BMC Microbiology 2004 4: 6
Predictions For Results
• There will be a significant difference in communities between
specialized & non-specialized feeders
• Intraspecific analysis will show less variability than analysis
interspecifically
• Community analysis of growth in medium will show less diversity
along a gradient with known solanine presence
Acknowledgements
Dr. Cristina Vesbach
Dr. Eric Toolson
Jennifer Johnson
NSF & UNM Sevilleta LTER
Scott Collins, John Dewitt,
Don Natvig
U.S. Fish & Wildlife
All of the REUs who helped
throughout the summer:
especially Emerson Tuttle,
Ash Schafer, Damon Lowery,
Ashley Melendez & Emily Stinson.
Questions?